1. Field of the Invention
The present invention relates to the fields of molecular biology, immunology and medicine. More specifically, the invention relates to immunostimulatory oligonucleotides and therapeutic uses thereof.
2. Summary of the Related Art
The immune system has evolved to specifically recognize DNA that contains an unmethylated CpG dinucleotide motif, which commonly occurs in the DNA of pathogens such as bacteria and viruses. As a result, unmethylated CpG-containing DNA is potent stimulator of the vertebrate immune system. First reports of immune stimulation by DNA came from studies using bacterial DNA and short fragments of DNA containing palindromic sequences, both of which were double-stranded structures with phosphodiester backbones Tokunaga, T., et al., (J. Natl. Cancer Inst. 72: 955-962 (1984)) demonstrated potent anti-tumor activity for DNA isolated from Mycobacterium bovis BCG. Kataoka, T, et al., (Jpn. J. Cancer Res. 83: 244-247 (1992)), Hartmann et al. (European Journal of Immunology 33:1673-1641 (2003)), Marshall et al. Journal of Leukocyte Biology 73:781-792 (2003) showed a similar type of anti-tumor activity for synthetic oligodeoxynucleotides, the design of which was based on Mycobacterium bovis BCG cDNA sequences.
Sato, Y, et al., (Science 273: 352-354 (1996)) showed the importance of CpG-containing DNA in the application of DNA vaccines (see also Gurunathan S., et al. (Annu. Rev. Immunol. 18: 927-974 (2000)). Pisetsky, D. S., et al., (Mol. Biol. Rep. 18: 217-221 (1993)) and Krieg, A. M., et al., (Nature 374: 546-549 (1995)) showed that DNA containing unmethylated CpG-dinucleotides in specific sequence contexts (CpG DNA) activated the vertebrate immune system, leading to proliferation of B cells and activation of macrophages, monocytes, NK cells, and dendritic cells. In response to CpG DNA activation, immune cells secrete a number of cytokines including IL-12, IFN-γ, INF-α, IL-6 and TNF-α and express several co-stimulatory molecules (for example, see Pisetsky, D. S., et al. and Krieg, A. M., et al., supra).
Kandimalla, E. R., et al., (Curr. Opin. Mol. Ther. 4 122-129 (2002)) indicate that the presence and position of a CpG-dinucleotide and the sequences that flank it are critical for immunostimulatory activity. Agrawal, S., et al. (Current Cancer Drug Targets 1: 197-209 (2001)) discloses significant effects due to ribose modifications in the flanking sequences of the CpG oligonucleotides. These effects depend on the position and nature of substituents, including 2′-O-methoxyethoxy and 2′- or 3′-O-methyl groups. Yu, D., et al. (Bioorg. Med. Chem. 9: 2803-2808 (2001)) demonstrate that phosphate modifications can also increase or decrease immunostimulatory activity depending on their position. Yu D., et al. (Bioorg. Med. Chem. Lett. 11: 2263-2267 (2001)) and Yu D., et al. (Bioorg. Med. Chem. 11: 459-464 (2003)) disclose that activity can be increased by deletion of certain nucleobases. In addition Yu D., et al. (Bioorg. Med. Chem. 11: 459-464 (2003)) disclose that immunostimulatory activity can be increased by substitution of certain flanking nucleotides with non-nucleotidic linkers.
Yu D., et al. (Bioorg. Med. Chem. Lett. 10: 2585-2588 (2000)), Yu D., et al. (Nucleic Acids Res. 30: 4460-4469 (2002)), Yu D., et al. (Biochem. Biophys. Res. Commun. 297: 83-90 (2002)), Bhagat L., et al. (Biochem. Biophys. Res. Commun. 300: 853-861 (2003)) and Kandimalla E. R., et al. (Nucleic Acids Res. 31: 2393-2400 (2003) previously have shown that the 5′-terminus is involved in receptor recognition and that accessibility of this end is critical for activity. Kandimalla E. R., et al. (Bioconj. Chem. 13: 966-974 (2002)) disclose loss of immunostimulatory activity following 5′-terminal conjugation of ligands larger than fluorescein or a 5′-5′ linked dinucleotide. As 3′-conjugation is without effect, changes in uptake cannot account for the results (Id.). However, there have not been any systematic studies to elucidate the role of secondary structure of DNA on the resulting immune response. The invention herein provides information on immunostimulation by immunostimulatory DNA with 5′- and 3′-hairpin loops or sticky ends that can form duplexes.
The ability of immunostimulatory DNA to induce Th1 cytokine production and promote CTL responses with enhanced immunoglobulin production has been used for treating a broad spectrum of disease indications including cancers, viral and bacterial infections, inflammatory disorders and as an adjuvant in immunotherapy. Thus, the benefits of improving or modulating immunostimulatory DNA activity are clear, and there remains a need in the art to develop improved immunostimulatory nucleic acids.